This invention relates to human pancreatic elastase. More particularly, but not exclusively, this invention relates to human pancreatic elastase I, to DNA containing a base sequence coding for this elastase, to a host transformed by this DNA and to a process for preparing the elastase using the host.
Elastase is a serine protease, capable of hydrolyzing the fibrous insoluble protein known as elastin. Elastin is a scleroprotein forming connective tissues, tendons, aortic integuments and cervical bundles of higher animals. Elastin is only slightly degraded by other proteases such as pepsin and trypsin.
In the course of their study on arteriosclerosis, Balo' et al observed degradation of the elastin fibers of arterial walls, and postulated the presence of a degrading enzyme (Schweiz Z Pathol Bacteriol, 12, 350 (1949)). Subsequently, in 1952, Banga discovered an enzyme in the pancreas which specifically hydrolyses elastin. The enzyme was isolated in the form of crystals and named "elastase" (Acta Physiol Acad Sci Hung, 3, 317 (1952)).
Elastase has been confirmed to exist in the pancreas of most mammals, including humans, monkeys, cats, rabbits, etc. A correlation is recognized between elastase activity and the age of a human being: a marked lowering in elastase activity in the pancreas and plasma of males over 40 and of females over 60 years has been reported by Loeven and Baldwin (Gerontologia, 17, 170 (1971)).
In the case of patients with arteriosclerosis, the elastase activity in the pancreas was reported by Balo' and Banga to be markedly lower than that of healthy people, and in some cases it had completely disappeared (Nature, 178, 310 (1956)). Subsequent studies have also demonstrated that elastase not only catalyses the hydrolysis of elastin but also accelerates elastin biosynthesis.
Studies on the pharmacological action of elastase have been carried out in rats and rabbits, and have revealed the following effects:
1) inhibition of the deposition of lipids and calcium on arterial walls; PA1 2) removal of cholesterol and calcium from arterial walls; PA1 3) selective degradation of denatured elastin; PA1 4) acceleration of synthesis of elastin fibers in the arterial walls; PA1 5) lowering of serum lipids; and PA1 6) improvement of lipoprotein metabolism. PA1 1) restoration of elasticity and expandability of arterial walls; PA1 2) reduction of serum lipid abnormality; PA1 3) improvement of serum lipoprotein metabolism. PA1 (a) isolation of DNA from human tissue; PA1 (b) construction of a gene library by use of the isolated DNA; PA1 (c) from the gene library, isolation of a chromosomal gene which codes for human elastase I using as a probe a cDNA of an elastase I from another animal; PA1 (d) analysis of the genetic sequence and determination of the amino acid sequence of the human elastase I; and PA1 (e) chemical synthesis of DNA capable of coding for the amino acid sequence; or PA1 (f) instead of (d) and (e), synthesis of a cDNA on the basis of the mRNA which can be obtained by transferring of the chromosomal gene obtained in (c) into suitable animal cells. From the DNA, the corresponding protein can then be prepared, for example by the steps of: PA1 (1) inserting the DNA into an expression vector; PA1 (2) introducing the recombinant DNA in to a host organism; PA1 (3) culturing the host under conditions resulting in expression of the DNA sequence; and PA1 (4) isolating a compound including the protein.
In clinical studies conducted on the basis of the findings mentioned above, the following effects have become apparent after oral administration of elastase:
Elastase extracted and purified from porcine pancreas was used for the above studies. Two types of elastase ("elastase I" and "elastase II") exist in the porcine pancreas (Front Matrix Biol, 6, 1 (1978)). The elastase used in the studies was a mixture of elastase I and elastase II, with the former as the main component.
With the administration of porcine elastase to human beings, there is the risk of antibody formation due to the antigenic effect of the foreign protein. There is then the danger of anaphylaxis with repeated administration. Accordingly, human elastase is preferable for human use. However, it is extremely difficult to procure human elastase in sufficient quantities from the traditional source, the human pancreas.
At present, four kinds of human pancreatic elastase are known, human elastases IIA and IIB and human elastases IIIA and IIIB. It has been established that an elastase corresponding to porcine elastase I is little expressed in the human pancreas.
In general, in order to produce a desired protein by use of recombinant DNA technology and a host such as E. coli, the complementary DNA (cDNA) of the mRNA of the desired protein is synthesized and then transferred into the host. However, the mRNA for human elastase I is so little expressed in human pancreas that the corresponding cDNA is difficult to obtain.